Idiopathic (primary) pulmonary arterial hypertension (IPAH), a subgroup of the vascular injury-induced forms of pulmonary arterial hypertension (PAH), is a rare disorder associated with severe morbidity and high mortality rates. There are no routine screening tests or validated markers of disease activity in IPAH, or the broader group of PAH. Therefore, patients usually present at advanced stages of disease. The pathogenesis of IPAH and other forms of PAH remain unclear. Current thinking focuses on a two-hit hypothesis: 1) genetic susceptibility, and 2) a triggering stimulus that initiates pulmonary vascular injury, resulting in endothelial cell dysfunction. Endothelial cells are normally shed into the circulation and are a valuable source of clinical material for studying diseases characterized by endothelial cell dysfunction. Unfortunately, no clear methodology exists for isolating clinically relevant numbers of circulating endothelial cells (CECs). In the bench phase of the project we are using flow cytometry to develop a methodology for isolating clinically relevant numbers of viable CECs from healthy volunteers and patients with PAH. We hypothesize that CECs and/or peripheral blood mononuclear cells (PBMC) can be used to define a subset of differentially regulated biomarkers in IPAH and other forms of PAH that may lead to earlier diagnosis and better methods for measuring responses to therapy. We also hope to identify novel targets for future therapeutic interventions. In the clinical phase of the project, we recruited healthy volunteers and patients with IPAH and other forms of PAH (vascular injury induced pulmonary hypertension). Peripheral blood specimens were obtained for CECs and PBMCs for microarrays; the remaining plasma was saved for future application to cultured microvascular cells. A subset of subjects underwent right heart catheterization to assess pulmonary pressures and to obtain pulmonary blood specimens. We started actively enrolling into the protocol in June 2006. We enrolled 31 individuals prior to closing the protocol to enrollment in 2009. Preliminary data suggested that there was no trend towards CEC enrichment in pulmonary vein blood compared to peripheral blood (PB) for both the healthy volunteers (4.4 CEC/ml vs. 4.8 CEC/ml) and the PAH patients (2.4 CEC/ml vs. 3.0 CEC/ml). There was a trend towards CEC enrichment in pulmonary artery blood compared to PB for both the healthy volunteers (13.8 CEC/ml vs. 4.8 CEC/ml) and the PAH patients (3.3 CEC/ml vs. 3.0 CEC/ml). In 2010, total RNA for microarrays was prepared from PBMCs. To more fully characterize their transcriptome, 16 of these samples were further processed in 2011 for high density oligonucleotide microarray analysis. In addition to further study gene expression, in 2011, 24 plasma samples from healthy volunteers and patients with PAH were processed for application to cultured microvascular endothelial cells. An abstract based on the PBMC differential gene expression patterns in PAH was presented at the Annual American Thoracic Society Meeting in 2011. These patterns reflected both treatment related signatures and underlying disease pathophysiology. In 2011-12, peripheral blood mononuclear cells (PBMCs) from 10 patients with PAH were compared to matched controls. Circulating PBMCs in PAH were found to harbor gene expression changes that may reflect a response to persistent contact with an injured vascular bed. Thematic analysis associated this transcriptomic signature with inflammation, cell-to-cell signaling and interaction, cytoskeletal rearrangement, cellular movement, hemostasis and cell death. Spironolactone, a mineralocorticoid (MR) and androgen receptor (AR) antagonist, was found in vitro to suppress selected genes upregulated in patients with PAH. In addition in 2011 -12 we continued to develop a bioassay assessing global transcriptomic changes induced by plasma from PAH subjects compared to healthy controls using Affymetrix oligonucleotide microarrays. Exposure of human PAECs to plasma from 5 PAH subjects compared to 5 age, gender and race matched controls, identified over 300 differentially expressed transcripts at a 10% false discovery rate. Future experiments will utilize stored plasma currently available from PAH patients and healthy controls to examine the effects of circulating mediators on gene expression in BMPR2-deficient PAECs. The protocol remains open for further data processing, analysis, and manuscript preparation.